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Sordarin bound eEF2 unlocks spontaneous forward and reverse translocation on CrPV IRES

View ORCID ProfileZheren Ou, Alexey Petrov
doi: https://doi.org/10.1101/2023.03.04.530920
Zheren Ou
1Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA
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Alexey Petrov
1Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA
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  • For correspondence: petrov@auburn.edu
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ABSTRACT

The Intergenic Region Internal Ribosome Entry Sites (IGR IRESs) of Discistroviridae promote protein synthesis without initiation factors, with IRES translocation by elongation factor 2 (eEF2) being the first factor catalyzed reaction. Here, we developed a system that allows for the observation of intersubunit conformation of eukaryotic ribosomes at the single-molecule level. We use it to follow translation initiation and subsequent translocation of the cricket paralysis virus IRES (CrPV IRES). We observed that pre-translocation 80S-IRES ribosomes spontaneously exchanged between non-rotated and semi-rotated conformations but predominantly occupied a semi-rotated conformation. In the presence of eEF2, ribosomes underwent forward and reverse translocation. Both reactions were eEF2 concentration dependent, indicating that eEF2 promoted both forward and reverse translocation. The antifungal sordarin, stabilizes eEF2 on the ribosome after GTP hydrolysis in an extended conformation. 80S-CrPV IRES-eEF2-sordarin complexes underwent multiple rounds of forward and reverse translocations per eEF2 binding event. In the presence of sordarin, GTP hydrolysis or phosphate release were not required for IRES translocation. Together, these results suggest that in the presence of sordarin, eEF2 promotes the mid and late stages of CrPV IRES translocation by unlocking ribosomal movements, with mid and late stages of translocation being thermally driven.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 05, 2023.
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Sordarin bound eEF2 unlocks spontaneous forward and reverse translocation on CrPV IRES
Zheren Ou, Alexey Petrov
bioRxiv 2023.03.04.530920; doi: https://doi.org/10.1101/2023.03.04.530920
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Sordarin bound eEF2 unlocks spontaneous forward and reverse translocation on CrPV IRES
Zheren Ou, Alexey Petrov
bioRxiv 2023.03.04.530920; doi: https://doi.org/10.1101/2023.03.04.530920

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